Abstract: The specific aims of the ongoing work are : 1) to understand how p300 and CBP participate in the operations of hematopoietic stem cells and, as such, to learn how they operate as hematopoeitic tumor suppressing elements; 2) to search for and evaluate the biological effects of lead small molecule compounds that can effectively interfere with the interaction of p300/CBP CHl and the TAD-C domain of hypoxia -inducing factor and thereby act as anti-tumor agents; 3) to determine whether the E3B4 ubiquitin ligase function of p300/CBP represents a useful anti-cancer therapeutic target in MDM2 overexpressing human tumor cells and 4) to decipher the biochemical functions of TRRAP and p400 and to determine how a suitable perturbation of their function[unreadable] translates into an E1A and SV40 T Ag-dependent neoplastic phenotype as well as the tumorigenic properties of c-myc overexpressing human tumor cells.[unreadable] Aiml: Understanding how CBP and p300 operate in hematopoietic stem cell behavior and as hematopoietic tumor suppressors: Currently, we have succeeded in reconstituting CBP 4-ES cells with a wt copy of CBP and are generating chimeric animals with these ES cells vs mock reconstituted CBP -/- ES cells. The question at hand is whether the reconstituted CBP-/- ES cells give rise to hematopoetic progeny cells that populate the peripheral blood. CBP -/- cells do not do so, as noted in the Report and in Dr. Rebel?s recent PNAS paper. If this is the case and mock reconstituted CBP -/- ES cells behave as expected, this would clearly show that the defect in CBP -/- HSC is a property of CBP deficiency. If this is the case, we would then try to generate transfected[unreadable] pools of CBP-/- ES cells that each stably produces a defined internal dl mutant CBP species which is stable, nuclear, and lacks a given CBP domain. We have generated a series of dl mutant alleles that, in the aggregate, affects all known CBP functional domains. The underlying question is whether certain CBP domains must be intact in order to support normal HSC self-renewal activity and, if so, which. From such an analysis, we hope to identify the critical CBP structures, which is a necessary first step in determining which specific CBP-based function(s) is/are required for both normal HSC[unreadable] self renewal activity and for CBP tumor suppressing activity. Success in this endeavor may make it possible to determine whether these two functions are inextricably linked.[unreadable] Aim2: Using chemical biology to identify molecules that specifically target HIF-1-p300/CBP complexes and to understand their biological effects in hypoxic tumor cells: We are actively searching for small molecule compounds-together with colleagues at the Novartis Functional Genomics Research Institute-that can selectively perturb p300 CHl/HIF complex formation and HIF-1 coactivation in vivo by p300/CBP. From among the candidates, we hope to identify any that have selective anti-tumor activity in animals in tumors in which there is clear evidence of hypoxia and elevated HIF transcription activity. Understanding how such compounds elicit their anti-tumor activity in vivo would be a high priority. For the most active of those compounds, we would also attempt to search for any that form stable complexes with TAD-C, p300 CH1, and/or CHl/TAD-C complexes. If feasible, these small molecule- containing complexes would then be subjected to detailed NMR analysis (in collaboration with our colleague, Mike Eck) in search of a detailed structure that could provide insight into how such a compound interferes mechanistically with p300/HIF coactivation. [unreadable] Aim 3: Exploring the biological and clinical significance of p300/CBP E4 ubiquitin ligase function: In search of insight into the clinical cancer-related significance of p300/CBP-+p53 E4 polyubiquitination function, we are beginning to ask whether thioredoxin or Gal 4 DBD fusion proteins containing large segments or all of p300 CH1 that block the interaction of MDM2/p53 complexes with intact p300, vs identical segments containing small deletions or missense changes and lack blocking activity, when suitably expressed in multiple MDM2- overexpressing human tumor cells lines, interfere with their ability to form tumors in nude mice. If the wt version of one or[unreadable] more of these peptides blocks tumor formation and the mutant(s) do not, the next question is whether the same wt peptides have an anti-tumor effect in non MDM2 overexpressing human tumor cell lines of the same pathological histotype (e.g. gliomas, certain sarcomas). Similarly, we are beginning to search systematically for others ubstrates of p300/CBP E4 activity. Amongth e candidates are each of the known CH1 interacting proteins (HIF-la, stat2 ets 1, MDM2, p35srj etc). Interestingly all of these proteins are particularly unstable, and one wonders whether stability control is mediated, at least in part, by the E3/E4 activity of p300/CBP. [unreadable] Aim 4: Understanding the contribution of ElMSV40 T Ag/c-myc perturbation of p400/TRRAP function to their transforming functions: We are continuing to search for evidence the reveals the specific biochemical functions of TRRAP and p400. Moreover, given the apparent necessity that TAP 54alP remain bound to the p400 complex for c-myc to express its transforming function and that this heterodimer be eliminated from the complex for ElA to express its transforming function, we are engaged in a combined genetic and chemical biology approach to the analysis of the biochemical and the biological functions of this protein pair. For example, in one set[unreadable] of experiments we are utilizing active site- directed missense mutants of TAP 54 a to further test the hypothesis that it must remain both bound to the p400 complex and remain enzymatically intact for c-myc to serve its transforming function. In addition, in vivo imaging assays for the p400 complex perturbing effects of ElA, T Ag, and c-myc are being developed, using Drosophila polytene chromosomes where these complexes (highly homologous in subunit composition to the human case and the subunit proteins are clear orthologs oftheir human counterparts in each case) form specific stripes at defined locations. Ideally, the introduction of a suitable p400 binding segment of the[unreadable] relevant oncoprotein will perturb the localization andor the subunit composition of the stripes. If valid and perfected, such an assay has the potential to reveal the nature of, oncoprotein- induced functional perturbations of individual proteins in the complex in close to real time.[unreadable]